Cancer is the second leading cause of death in the United States. An estimated 10.1 million Americans are living with a previous diagnosis of cancer. In 2002, over one million people were newly diagnosed with cancer in the United States (information from Centres for Disease Control and Prevention, 2004 and 2005, and National Cancer Institute, 2005). According to Cancer Research UK, in 2005 over 150,000 people died in the UK as a result of cancer. It has been reported that almost 44,100 cases of breast cancer are diagnosed in the UK each year (i.e. 16% of all new cancer cases), and over 12,400 UK breast cancer patients die annually from this disease (approx. 8% of all deaths caused by cancer). One of the most common cancers is colon cancer, with approximately 21,600 new cases diagnosed in the UK during 2003. Colon cancer is also one of the most common causes of death from cancer, and it accounted for approximately 10% of all cancer-related deaths in 2005.
There is clearly a need to develop improved methods for diagnosis and treatment of cancers in the UK, the US and throughout the world.
Cancer is caused by the mutation of genes or the (change of) expression of genes that trigger uncontrolled cell division. There are two types of genes which when mutated may lead to cancer: (i) proto-oncogenes, which promote cell growth and mitosis, i.e. cell division (for example, mdm2 or the human homologue, hdm2); and (ii) tumour suppressor genes, which inhibit cell growth or halt cell division (for example, p53 and p21).
Proto-oncogenes often produce mitogens, which encourage cell division; or they may be transcription factors, transcription co-repressors or transcription co-activators, which can increase or change the expression of other genes. In particular, proto-oncogenes may encode proteins that regulate the expression or activity of tumour suppressor proteins. Viruses are another source of (proto-)oncogenes in humans, and it is thought that approximately 15% of human cancers worldwide originate from viral infections. The main viruses associated with human cancers are human papillomavirus, hepatitis B virus, Epstein-Barr virus, human T-lymphotropic virus and Kaposi's sarcoma herpesvirus.
Tumour suppressor genes typically encode anti-proliferation molecules that suppress mitosis and cell growth. Often, tumour suppressor proteins are also transcription factors, but their expression tends to be activated by cellular stress or DNA damage. Tumour suppressors generally function to arrest cell cycle progression, thereby giving the endogenous cellular processes time to repair any damaged DNA (resulting from the cellular stress) and prevent the multiplication of DNA mutations. Alternatively, for example, where a cell has been exposed to extreme levels of stress with little chance of recovery, a tumour suppressor may trigger apoptosis of the affected cell. Hence, where the expression of a tumour suppressor gene has been inhibited, DNA mutations are able to accumulate unchecked and, almost inevitably, will lead to cancer. The most common tumour suppressor gene associated with cancer is p53, which has been reported to display an altered expression profile and/or mutations in almost half of all cancers.
Detecting cancer at an early stage in the development of the disease is a key factor in enabling the disease to be effectively treated and prolonging the life of the affected individual. Cancer screening is an attempt to detect (undiagnosed) cancers in the population, so as to enable early therapeutic intervention. Screens for detecting and/or predicting cancer are advantageously suitable for testing large numbers of subjects; are affordable; safe; non-invasive; and accurate (i.e. exhibiting a low rate of false positives).
A number of different screening tests have been developed, many of which involve visual/tactile examination, e.g. for breast or testicular cancer. However, many cancers cannot be detected by such crude examinations and, in these cases, other forms of physical and/or biochemical examinations may be required. For instance, screening by mammogram can detect a breast tumour earlier than self-examination; colorectal cancer can be detected, for example, through faecal occult blood testing and colonoscopy; and cervical cytology testing (smear test) can lead to the identification of precancerous lesions. Meanwhile, a screen for prostate cancer uses a digital rectal exam along with a blood test to detect prostate specific antigen (PSA).
There remains, however, a large number of different types of human cancer, which are not readily detectable, or reliably detectable, by present screening methods, prior to the onset of disease symptoms (by which time it may be too late for an effective intervention). Accordingly, there is a need for further cancer detection/screening methods, particularly biochemical methods, which will allow the detection of cancer before the onset of disease symptoms. There is also a need for additional screens to supplement and/or confirm the diagnoses from cancer screens already known in the art.
The p53 tumour suppressor is a key component of the cellular processes that maintain genomic integrity in response to cellular stress and/or DNA damage (Levine, 1997, Cell, 88(3): 323-331) and, as already noted, aberrations in p53 are associated with over half of all human cancers. Under normal conditions, p53 and its cellular activity are maintained at low levels by a combination of rapid degradation and inhibition, both of which activity regulation processes are thought to be largely controlled by the product of the mdm2 (or hdm2 in humans) proto-oncogene. The hdm2 protein has been shown to bind to the transactivation domain of p53 and inhibit its transactivation activity (Momand et al., 1992, Cell, 69(7): 1237-1245). In addition, it has been reported that hdm2 functions as an E3 ubiquitin ligase (Honda at al., 1997, FEBS Lett., 420), which acts on p53 to target it for proteasomal degradation via a ubiquitin-dependent pathway (Haupt at al., 1997, Nature (London), 387: 296-299; Kubbutat at al., 1997, Nature (London), 387: 299-303). Significantly, the expression of the hdm2 gene is itself controlled by p53 and, therefore, both the cellular concentration and the activity of p53 are under the control of an auto-regulatory feedback loop involving hdm2.
As a result of cellular stress, for example, DNA damage induced by UV radiation or mutagens, p53 is transiently stabilised (by phosphorylation in its N-terminal domain), which prevents the binding of hdm2 and stabilises its active conformation. Once activated, p53 induces the transcription of a number of genes, including p21, bax and hdm2 (as previously described). Both p21 and bax proteins mediate the anti-proliferative function of p53 by blocking cell cycle progression and/or provoking cell apoptosis (El-Deiry at al., 1995, Cancer Res., 55: 2910-2919; Miyashita & Reed, Cell, 80(2): 293-299). The tumour suppressor protein p21 (also known as “cyclin-dependent kinase inhibitor 1A” or “CDKN1A” because of its mode of action), plays an important role in mediating cell cycle arrest in the G1-, G2- or S-phase when cellular DNA is damaged. It has been demonstrated that both p21 and p53 are essential for stabilising the G2 checkpoint after DNA damage in human cells (Bunz at al., 1998, Science, 282: 1497-1501).
That p53 is associated with the onset and/or progression of so many cancers, makes it a prime target for regulation or modification for cancer therapy. In addition, the genes and/or proteins that are activated or repressed by p53, and conversely, those that are responsible for controlling the activation or repression of p53 may also be useful targets for anti-cancer drugs and treatments.
One p53-interacting protein is hdm2 (as previously discussed): another is TRIM28 (tripartite motif-containing 28, also known as KAP1, Krip1, RNF96, TF1B, TIF1B, TIF1 beta, TIF1β, UniProtKB/Swiss-Prot entry Q13263), which is a member of the family of proteins that contains a TIF1 domain structure. The TIF1 domain comprises an N-terminal region having an RBCC (RING finger-B boxes-coiled coil) motif; a poorly conserved central region; and a C-terminal region containing a PHD finger and a bromodomain or “BROMO” (see for example, Le Douarin et al., 1996, EMBO J., 15: 6701-6715). TRIM28 has been identified almost exclusively as a non-enzymatic component of transcriptional regulatory complexes (Friedman et al., 1996, Genes Dev., 10: 2067-2078; Moosmann et al., 1996, Nucleic Acids Res., 24: 4859-4867). Although not believed to bind DNA itself, TRIM28 is known to interact with a wide variety of nuclear factors including KRAB repressors (Kim et at., 1996, Proc. Natl. Acad. Sci. USA, 93: 15299-15304); the histone-binding protein HP1; SETDB1 (ESET); and hdm2 (Wang et al., 2005, EMBO J., 24: 3279-3290). In fact, TRIM28 is thought to be a member of several larger multi-factor complexes that typically function in the repression of gene expression and the stabilisation of heterochromatin (Sripathy et al., 2006, Mol. Cell. Biol., 26: 8623-8638).
Other members of the TRIM family include: TRIM25 (also known as EFP, RNF147, Z147 and ZNF147), in which the RING domain has been shown to possess E3 ubiquitin ligase activity that is important in mediating anti-viral activity (Gack, et al., 2007, Nature, 446: 916-920); and TRIM33 (also known as PTC7, RFG7, TF1G, TIF1G, FLJ32925, TIFgamma, TIF1gamma and TIF1γ), which also has been shown to exhibit E3 ubiquitin ligase activity via its RING domain (Dupont et al., 2005, Cell, 121: 87-99).
Another member of the TRIM family, that is also a member of the TIF1 protein sub-family, is TRIM24 (also known as PTC6, TF1A, TIF1, RNF82, TIF1A, hTIF1, TIF1ALPHA and TIF1α), which is known to interact with certain nuclear receptors and to possess a kinase activity (Fraser et al., 1998, J. Biol. Chem., 273: 16199-16204). More specifically, TRIM24 has been shown to mediate transcriptional control by interacting with the AF2 (activation function 2) activating domain of selected nuclear receptors (NRs) including the oestrogen, retinoic acid and vitamin D receptors (Thenot et al., 1997, J. Biol. Chem., 272(18): 12062-12068). In this study it was suggested that TRIM24 may regulate transcription by a mechanism of chromatin remodelling, rather than through the transcriptional machinery. TRIM24 has also been shown to interact with heterochromatin-associated proteins (Le Douarin of at., 1996, EMBO J., 15: 6701-6715). Another recent publication has indicated that TRIM24 is involved in the modulation of gene expression during the first transcriptional wave at the zygote (1-cell) stage (Torres-Padilla & Zernicka-Goetz, 2007, J. Cell Biol., 174: 329-338), which further suggests a possible epigenetic role for TRIM24.
There have been no previous reports of an interaction between TRIM24 and p53, or evidence for the direct involvement of TRIM24 in p53-associated cancers. Similarly, there is no reported evidence for an E3 ligase activity for TRIM24 (c.f. TRIM25 and TRIM33).
The present invention seeks to overcome or at least alleviate some of the aforementioned problems in the art, for example, first by identifying factors that may be involved in disease states such as cancer, and particularly, cancers that are associated with p53 alterations; and then by identifying modulators of p53 activity for treating cancer.